Photoelectrochemical Synthesis of High Density Combinatorial Polymer Arrays

ABSTRACT

In a method for creating polymer arrays through photoelectrochemically modulated acid/base/radical generation for combinatorial synthesis, electrochemical synthesis is guided by a spatially modulated light source striking a semiconductor in an electrolyte solution. A substrate having at its surface at least one photoelectrode that is proximate to at least one molecule bearing at least one chemical functional group is provided, along with a reagent-generating chemistry co-localized with the chemical functional group and capable of generating reagents when subjected to a potential above a threshold. An input potential is then applied to the photoelectrode that exceeds the threshold in the presence of light and that does not exceed the threshold in the absence of light, causing the transfer of electrons to or from the substrate, and creating a patterned substrate. The process is repeated until a polymer array of desired size is created.

RELATED APPLICATIONS

This application is a continuation of U.S. Provisional application Ser.No. 11/698,282, filed Jan. 25, 2007, which claims the benefit of U.S.Provisional Application Ser. No. 60/761,844, filed Jan. 25, 2006, theentire disclosures of which are each herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under Grant NumberCCR-0122419, awarded by the National Science Foundation. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to creation of polymer arrays and, in particular,to generation of polymer arrays by photoelectrochemical patterning.

BACKGROUND

In recent years, hybridization arrays [e.g., Fodor S P, R. J., Pirrung MC, Stryer L, Lu A T, Solas D. (1991), “Light-directed, spatiallyaddressable parallel chemical synthesis”, Science 251(4995): 767-73] andon-chip sequencing-by-synthesis [e.g., Quake, E. P. K. a. S. R. (2004),“Microfluidic device reads up to four consecutive base pairs in DNAsequencing-by-synthesis”, Nucleic Acids Research 32(9): 2873-2879] haverevolutionized genome sequencing because of their massively parallelgenomic sequencing power. Several techniques for in-situ DNA arraysynthesis [e.g., U.S. Pat. Nos. 6,054,270; 5,700,637] have been reportedin the literature: photocleavable 5′ and 3′ protecting groups [Fodor(1991);U.S. Pat. Nos. 5,445,934, 5,744,305, 5,677,195; Singh-Gasson, S.,R. D. Green, et al. (1999), “Maskless fabrication of light-directedoligonucleotide microarrays using a digital micromirror array.” NatureBiotechnology 17(10): 974-978], deprotection of the acid labile tritylgroups by photogenerated acids (Gao, X., E. LeProust, et al. (2001), “Aflexible light-directed DNA chip synthesis gated by deprotection usingsolution photogenerated acids”, Nucleic Acids Research 29(22):4744-4750], and electrochemical acid generation [Dill, K., D. D.Montgomery, et al. (2004), “Immunoassays and sequence-specific DNAdetection on a microchip using enzyme amplified electrochemicaldetection”, Journal of Biochemical and Biophysical Methods 59(2):181-187; U.S. Pat. No. 6,093,302; Egeland, R. D. and E. M. Southern(2005), “Electrochemically directed synthesis of oligonucleotides forDNA microarray fabrication”, Nucl. Acids Res. 33(14): e125], ink jettingreagents [Hughes, T. R., M. M., Jones A R et al. (2001), Nat. Biotech.4: 342-347], and microcontact printing [Xiao, P. F., N.Y. He, et al.(2002), “In situ synthesis of oligonucleotide arrays by using softlithography”, Nanotechnology 13(6): 756-762].

Chips synthesized using photocleavable protecting groups havedemonstrated high spot densities, but the reagent costs for suchphosphoramidite reagents are currently prohibitively high fornon-commercial laboratories (typically more than $300/g). The UVexposure deprotection times are also quite long, usually on the order ofminutes. Methods that locally photogenerate acids to detritylatestandard phosphoramidite reagents do offer improved deprotection times[Gao (2001)], but they have yet to show the same spot density as thosegenerated using photocleavable protecting groups, presumably because ofacid diffusion. Systems based on electrochemical acid generation havealso demonstrated quick deprotection times, but circuitry required tomake individually addressable electrodes limits is costly to fabricateby photolithographic means [e.g., U.S. Pat. No. 6,093,302]. Extremelyhigh-resolution chips have been created by micro-contact printing ofphosphoramidites, but this technique is limited in its versatilitybecause it requires the creation of a new stamp for each base added[Xiao (2002)].

A low-cost and rapid synthesis platform of high-resolution DNA chipswould be of great utility to laboratory scientists. Such a platformwould open up many other exciting possibilities in biological research;for example, it has been recently shown that genomic length DNA can beassembled from the short oligonucleotides from such chips. Furthermore,the techniques used to create DNA chips can be used to pattern otherbiomolecules [Shuwei Li, D. B., Nishanth Marthandan, Stanley Klyza,Kevin J. Luebke, Harold R. Garner, and Thomas Kodadek (2004),“Photolithographic Synthesis of Peptoids”, J. Am. Chem. Soc. 126(13):4088-4089] and polymers on surfaces. What has been needed, therefore, isa means for quickly and inexpensively creating polymer arrays.

SUMMARY

The present invention is a method for creating inexpensiveoligonucleotide, protein, or other polymer arrays throughphotoelectrochemically modulated acid/base/radical generation forcombinatorial synthesis, where electrochemical synthesis is guided by aspatially modulated light source striking a semiconductor in anelectrolyte solution. A semiconducting device is in contact with anelectrolyte solution, matrix, gel, or solid that is suitable as aplatform for electrochemical reactions at a surface. Light patterned bya mask, LED, LCD, steered mirror, or digital micromirror array is usedto generate charge carriers in the semiconductor, which then generateelectrochemical reactions via direct transfer of electrons to or fromthe semiconductor, optionally through a metal or mediator, thus allowingfor spatially-guided electrochemistry.

In one aspect, the present invention is a method to immobilize and/orin-situ build biomolecule, bio-polymer, small molecule, and polymerarrays by photoelectrochemical patterning. In particular, the presentinvention may be employed to fabricate DNA arrays by phosphoramiditesynthesis.

In another aspect, the present invention is a method forphotoelectrochemical placement of a material at a specific location on asubstrate. A substrate having at its surface at least one photoelectrodethat is proximate to at least one molecule bearing at least one chemicalfunctional group is provided, along with a reagent-generating chemistryco-localized with the chemical function group and capable of generatingreagents when subjected to a potential above a threshold. An inputpotential is then applied to the photoelectrode that exceeds thethreshold in the presence of light and that does not exceed thethreshold in the absence of light, causing the transfer of electrons toor from the substrate. In one embodiment, the chemical functional groupis protected and the generated reagents are deprotecting. In anotherembodiment, the chemical functional group is unprotected and thegenerated reagents are activating. In a further embodiment, theprotected chemical functional group is located on a second parallelsubstrate and the reagent-generating chemistry can diffuse towards theprotected chemical function group on the second substrate.

In a further aspect, the present invention is a method forphotoelectrochemical synthesis of a polymer array, comprising the stepsof providing a substrate having at the substrate surface at least onephotoelectrode that is proximate to at least one molecule bearing atleast one chemical functional group, providing a reagent-generatingchemistry co-localized with the chemical function group capable ofgenerating reagents when subjected to a potential above a threshold,applying an input potential to the photoelectrode that exceeds thethreshold in the presence of light and that does not exceed thethreshold in the absence of light, and repeating until a polymer arrayof desired size is created.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing discussion will be understood more readily from thefollowing detailed description of the invention, when taken inconjunction with the accompanying drawings, in which like referencedfeatures identify common features in corresponding drawings and:

FIG. 1 is a schematic of an embodiment of a system employed forphotoelectrochemical synthesis of polymer arrays, according to oneaspect of the present invention;

FIG. 2 illustrates the steps in two embodiments of a method for thephotoelectrochemical placement of a material at a specific location on asubstrate to create a photoelectrode substrate, according to one aspectof the present invention;

FIG. 3 is an Scanning Electron Microscope micrograph of patternedplatinum microcontacts on amorphous silicon over ITO-glass, as createdaccording to one aspect of the present invention;

FIGS. 4A and 4B are Atomic Force Microscopy and Scanning ElectronMicroscope micrographs, respectively, of a porous SiO2 reaction layercreated from sintered commercial colloidal silica, according to oneaspect of the present invention;

FIG. 5 is a schematic of an embodiment of a DMD-based spatiallymodulated illumination system used to drive site-selectivephotoelectrochemistry, according to one aspect of the present invention;

FIG. 6 is a photoelectrochemical cyclic voltammogram of ferrocenedemonstrating selective photo-induced redox chemistry; and

FIG. 7 is a fluorescence micrograph of a dye that has selectivelyreacted with functional groups that are only photoelectrochemicallydeprotected over illuminated electrodes, according to one aspect of thepresent invention.

DETAILED DESCRIPTION

The present invention is a method and apparatus for creating inexpensivenucleotide, protein, or other polymer arrays throughphotoelectrochemically modulated acid/base/radical generation forcombinatorial synthesis, where electrochemical synthesis is guided by aspatially modulated light source striking a semiconductor in anelectrolyte solution. A semiconducting device is in contact with anelectrolyte solution, matrix, gel, or solid suitable as a platform forelectrochemical reactions at a surface. Light patterned by a mask, LED,LCD, steered mirror or digital micromirror array is used to generatecharge carriers in the semiconductor, which then generateelectrochemical reactions via direct transfer of electrons to or fromthe semiconductor, or through a metal or mediator, thus allowing forspatially guided electrochemistry. As all addressing occurs off of thedevice, one skilled in the art can recognize and appreciate thatsubstrate costs are minimal.

In one aspect, the present invention comprises a method to immobilizeand/or in-situ build biomolecule, bio-polymer, small molecule, andpolymer arrays by photoelectrochemical patterning. In particular, thepresent invention permits the fabrication of DNA arrays byphosphoramidite synthesis. This technique has been used to pattern andmove biomolecules and polymer beads, to move fluids in microfluidics byopto-electrowetting (Chiou, P. Y., H. Moon, et al. (2003), “Lightactuation of liquid by optoelectrowetting.” Sensors and Actuators, A:Physical A104(3): 222-228), and to control electrochemistry for analytedetection (Hafeman, et al. (1988), “Light-Addressable PotentiometricSensor for Biochemical Systems”; George, M., W. J. Parak, et al. (2000),“Investigation of the spatial resolution of the light-addressablepotentiometric sensor”, Sensors and Actuators A: Physical 86(3):187-196; Tatsu Yoshinobu, et al. (2004), “Fabrication of Thin-Film LAPSwith Amorphous Silicon”; U.S. Pat. No. 6,682,648). Because theelectrodes are light addressable, there are no physically addressableelectrodes required, and thus the fabrication of such a device is cheapand facile, while still offering high resolution or spot density.Reagent costs are low as the platform relies on standard phosphoramiditereagents. The present invention therefore allows DNA and polymer arraysto be manufactured more cheaply and faster than with current alternativemethods.

In a typical embodiment, the device is held in a fluidic capable ofelectrochemical synthesis and chemical resistance to solvents, acids,and bases. Nickel or nickel alloys, glass, fluorinated hydrocarbons(such as Teflon), fluorinated elastomers, high density polyethylene orpolypropylene, gold, and platinum group metals (PGMs) are examples ofsuitable materials, but other materials may also be advantageouslyemployed in the present invention. Teflon, glass, and platinum arepreferred for their inertness to electrochemical processes. The fluidicmust support the connections to the semiconducting device electrode andmaintain the counter electrode in contact with the electrolyte. Thefluidic must also have a window to the semiconductor surface, throughwhich the semiconductor is illuminated. This window may contact thesolution (“front side” illumination) so that light illuminates thesemiconductor through solution. It is preferable that the window also bea conductor, so that the surface area of the counter electrode is atleast as large as that of the semiconducting electrode surface. A clearconducting oxide is an example of a suitable material, and is evenbetter if a thin passivation layer of platinum, palladium, or iridium isapplied to the surface such that it still transmits enough light to thesemiconducting surface. If the window enables front-side illumination,it is possible to directly monitor the removal of the protecting groupor the generation of the protecting reagent in real-time. For example,UV-Vis absorbance measurements or imaging with a color-filtered CCDcamera may be employed, since the trityl protecting group absorbs at˜298 nm and a protected phosphoramidite is clear in solution, but thepost-deprotection cation absorbs light at ˜498 nm and the solution turnsorange. Alternatively, the window may directly contact the semiconductor(such as a glass/ITO window), which is known as “back side”illumination. In this manner, the ITO/glass serves as both a window andan electrical contact.

FIG. 1 is a schematic of an embodiment of a system forphotoelectrochemical synthesis of polymer arrays, according to oneaspect of the present invention. In the preferred embodiment of FIG. 1,the photoelectrochemistry fluidic for photoelectrochemical patterningcomprises fluidic chamber 100 that includes plastic compression fittings105, Teflon spacers 110 (glued via Viton), reference electrode 115(platinum quasi-reference), photoelectrode substrate 120, counterelectrode 125 (nickel fluidic), fluid inlet 130, fluid outlet 135,Kalrez O-rings 140, and window 145 for illumination from a light source150 (hv). FIG. 1 depicts a “back-side” illumination fluidic, as lightpasses through the substrate instead of through solution. Depending onthe system specifications, solution side (front side) or substrate side(back side) illumination may be preferable, in which case the devicepreparation and fluidic design is adjusted accordingly, as is well-knownin the art of the invention.

The photoelectrode substrate is preferably a semiconductor. Thesemiconductor may be intrinsic, n-type, p-type, or some multi-layerstructure such as a PIN photodiode operated in reverse bias. Intrinsicor n-type semiconductors, such as, but not limited to, TiO2, amorphousor crystalline silicon, zinc sulphide, and cadmium selenide, have betteretch resistance properties when under a positive bias with respect tothe electrolyte solution if they are to be in direct contact. Ingeneral, silicon is typically preferred, because the processing steps inpassivating the surface from electrochemical or chemical degradation areeasier.

The semiconductor may be covered with a protective material, such as,for example, an inert metal or a different semiconductor, in order totake advantage of the respective material properties. For example,amorphous silicon may be covered with mesoporous or flat titaniumdioxide, thereby creating a surface suitable for direct, stableelectrochemical reaction, but still having silicon dominate thephotoconductive gain. Alternatively, for example, amorphous silicon maybe covered with thin silicon nitride, which is known to prevent thedehydrogenation of amorphous silicon, thereby improving thephotosensitivity and lifetime of the photoconductor. The electrolytesolution can be aqueous, organic, or ionic, depending on thesemiconductor used and the desired reaction to take place.

In a typical embodiment, the semiconducting device is under theapplication of an electric field, either intrinsic to the device, suchas via a PIN structure, or externally applied, in order to guide thecharge carriers generated by illumination in a desired direction, butthe selection of semiconducting material or layers of material may besuch that photoexcited charge moving across the semiconductor spacecharge layer in contact with the solution has enough energy to reactdirectly with an electrolyte. Upon illumination with a specific lightpattern, areas under illumination will generate charge carriers, andthus be capable of electron capture from the solution to thesemiconductor and overall decreased impedance of the semiconductorlayer. In an alternate embodiment, upon illumination with a specificlight pattern, areas under illumination will generate charge carriers,and thus be capable of electron injection to the solution from thesemiconductor.

Whereas the applied bias potential drop is primarily across thesemiconductor in a non-illuminated semiconductor-electrolyte interface[Bard, A. J., Stratman, M. S., and Licht, S. Semiconductor Electrodesand Photoelectrochemistry. Wiley-VCH: Chapter 1], the potential drop isnearly entirely shifted to the double layer of the electrolyte when thephotoconductive electrode is illuminated with sufficient light becauseof the decreased impedance. Thus, non-illuminated electrodes are at muchlower potentials at the interface than the bias potential, butilluminated ones are effectively at the bias potential at the interface.This provides the contrast necessary to perform a desired reaction atonly the locations that are illuminated, since electrochemical reactionsare highly non-linear with respect to surface potential. Features assmall or smaller than a few microns are possible utilizing the presentinvention, demonstrating that the present invention compares withcurrent commercially available high-density arrays, while beingconsiderably cheaper to manufacture. In the preferred embodiment, thesurface is biased at or above the activation energy or thresholdvoltage, but still within the potential window of the electrolytesystem.

A typical device is fabricated with plasma enhanced vapor deposition(PECVD) of amorphous silicon onto a suitable conductive surface, such asindium tin oxide. A front side illuminated device may have a thin filmof amorphous silicon deposited onto steel or aluminum or any otherconductor with appropriate adhesion to silicon, or may be simply a waferof crystalline silicon with an Ohmic contact, such asevaporated/annealed gold. The front side illumination system ispreferable if there is low or nonexistent applied external bias or highdopant concentration, because more charge carriers are generated at thesurface and thus are less likely to recombine before reacting with theelectrolyte (as opposed to charge carriers generated in the bulk, whichmust first diffuse to a space charge region in the semiconductor). Abackside-illuminated device may have a thin film (typically 500 nm to 2um) of amorphous silicon deposited onto a clear conducting oxide, suchas indium tin oxide, on a flat glass slide (such as Corning 1737 Glassor polished float glass), which serves as the window to thesemiconductor. Front-side illumination systems enable the additional useof photocleavable and photogenerated chemistries. A combination of bothfront- and back-side illumination may also be employed.

In one embodiment, material is photoelectrochemically placed at aspecific location on a substrate by providing a substrate having at thesubstrate surface at least one photoelectrode that is proximate to atleast one molecule bearing at least one chemical functional group,providing a reagent-generating chemistry co-localized with the chemicalfunction group capable of generating reagents when subjected to apotential above a threshold, and applying an input potential to thephotoelectrode that exceeds the threshold in the presence of light andthat does not exceed the threshold in the absence of light. In anotherembodiment, a polymer array is synthesized by providing a substratehaving at the substrate surface at least one photoelectrode that isproximate to at least one molecule bearing at least one chemicalfunctional group, providing a reagent-generating chemistry co-localizedwith the chemical function group capable of generating reagents whensubjected to a potential above a threshold, applying an input potentialto the photoelectrode that exceeds the threshold in the presence oflight and that does not exceed the threshold in the absence of light,and repeating until a polymer array of desired size is created.

When the array is being constructed by selectivelyactivating/deprotecting a site and immobilizing pre-synthesizedmolecules, the reactive group can sit directly atop the electrode, anddirect photoelectrochemistry may be employed [e.g., Kim et al. (2002),Langmuir 18, 1460-1462]. This should typically be avoided for in-situsynthesized arrays, because electrochemical damage to the growingpolymer may occur. Adequate linking chemistry is necessary that canwithstand electrochemical processing, allowing for reagents to diffuseproperly and adequately bind the DNA to the substrate. This linkingchemistry can be broken down into two parts, with the first being thedevelopment of an insulating microporous reaction layer bound to thesurface of the chip that allows the growing DNA strand to be proximal tothe electrochemistry during the deprotection step but sufficiently farfrom the electrode (beyond the Helmholtz layer, ˜5-10 nm) to not incurelectrochemical damage. The electrochemistry may also generateactivating/catalytic chemistry instead of deprotecting reagents. Forexample, an acid created may be used to deprotect a dimethoxytritylgroup during a phosphoramidite synthesis as shown, or activate thephosphoramidite addition towards an already deprotected hydroxyl.

A porous reaction layer has the benefit of vastly increasing the surfacearea of the chip, thereby increasing the total crude product quantityyield (U.S. Pat. No. 6,824,866). In some cases, this layer can alsoincrease the crude product quality yield because of increased masstransport and decreased sterics (Zhou et al. Nucleic Acids Res. 2004,32, 18, 5409-5417). Data demonstrating the increased quantity andquality from a porous reaction layer is presented in Table 1, which is atable of surface loading capacities of DNA phosphormidites andas-synthesized oligonucleotides on the porous reaction layer accordingto one aspect of the present invention.

TABLE 1 40-mer oligonucleotide Step- Dimethoxytrityl cation Area densityConcentration Crude wise Area density Concentration (molecules/cm²) (mM)yield yield Substrate (molecules/cm²) (mM) Unpurified PurifiedUnpurified Purified (%) (%) Flat glass 1.8 ± 0.1 × 10¹⁴ N/A 3.1 ± 0.5 ×1.9 ± N/A N/A 59.4 ± 98.7 ± 10¹³ 0.3 × 3.4 0.1 10¹³ Porous 1.3 ± 0.2 ×10¹⁶ 25.0 ± 2.8 4.7 ± 0.2 × 2.4 ± 9.4 ± 0.4 4.7 ± 76.4 ± 99.3 ± oxide10¹⁴ 0.2 × 0.4 5.3 0.2 10¹⁴ CPG N/A 13.2 ± 0.3 N/A N/A 7.3 ± 0.1 5.9 ±85.5 ± 99.6 ± 0.3 5.2 0.2

The second part of the process of the present invention employs asuitable linking chemistry that binds the DNA to the microporousreaction layer. This includes, but is not limited to: alkenes forhydrosilylation to silicon, thiols and amines on metals, andorganosilanes, carboxylic acids, and phosphonic acids to metals, metaloxides, semiconductors, and semiconductor oxides. It is also possiblefor electrochemically-generated acid that diffuses from thesemiconducting substrate to react with molecules on the surface ofanother solid support. For example, the device can be inverted so thatthe reactive chemical species is in near proximity to the surface uponwhich DNA is synthesized, thereby allowing the device structure to bereused.

The microporous reaction layer may be composed of a wide range ofmaterials produced by a variety of methods. Such reaction layersinclude, but are not limited to, porous oxidized aluminum (via chemicalor electrochemical oxidation), porous silicon (via electrochemicaletching), a porous titanium dioxide layer made from solution phasenanoparticles or sol-gel processing, a porous polysilicon layer, aporous silicon dioxide layer made from colloidal silica, silanesol-gels, spin-on-glasses, a porous silicon dioxide formed bypost-deposition chemical or ion-etching, or the immobilization ofstandard solid-phase supports like controlled-pore glass (CPG) andMerrifield resins. Of these materials, silicon dioxide is preferable forthe facility of silane coupling and chemical inertness, though all ofthe materials mentioned are capable of suitable linking chemistry forfunctionalization. In the case of electrochemical acid diffusion toreact upon an alternative surface, a porous reaction layer is notnecessary, though it may be capable of generating improved total amountof product because of the increased surface area for linking chemistry.

The surface of a DNA microarray must have a suitable linking chemistryto attach a nascent strand of DNA and withstand the rigors of thephosphoramidite synthesis cycle. Silane functionalization is a widelyused technique for DNA microarrays because of their reactivity withglass surface hydroxyls and their stability. Fortunately, glass israther easy to functionalize with organo-silane linkers, although it hasbeen demonstrated that silanization is possible with a variety of metaloxide surfaces, including titania and alumina. Generally, a silaneprecursor, such as a hydroxyl or amino functionalized chlorosilane oralkoxysilane, is reacted with the free hydroxyls on an oxide surface andthen permitted to cross polymerize with heat or exposure to air to forma resistant polysilane monolayer. In a typical embodiment, treatmentwith triethoxysilane hydroxybutyramide or 3-aminopropyl triethoxysilaneprovides a free hydroxyl group or amine respectively, which are thenused for the attachment of further chemistries, such as, but not limitedto, phosphoramidites used in DNA synthesis. These are suitable linkingchemistries for surfaces with free hydroxyl groups, such as SiO2, TiO2,and most materials that have surface oxides. Alternatively, ahydrosilation reaction can be used to provide an attachment chemistry toan HF treated silicon surface, which has a hydrogen terminated surface.Suitable hydrosilation coupling compounds are generally a terminalalkene with a protected nucleophile at the opposing end.

Typically, the device must withstand the rigors of the chemicalprocessing and serve as a suitable electrode in an electrolyte.Therefore, a passivating surface may be added to the semiconductor tomake it more suitable to electrochemical processing. This film may be athin film of SiO2, such as the native oxide of a silicon wafer or thenative oxide developed onto a PECVD deposited amorphous silicon film, itmay be TiO2, or it may be silicon nitride, silicon carbide, siliconoxynitride, silicon carbonitride, tetrahedryl amorphous carbon, ornitrogen doped tetrahedryl amorphous carbon or other suitable materialsknown in the art.

Ideally, the passivating film is itself semiconducting, because thatenables the substrate fabrication to eliminate all patterning steps, asa thin chemically inert semiconductor would not contribute significantlateral electron currents or electrical impedance, especially if thesemiconductor were itself a photoconductor. Alternatively, the top layerof the semiconductor may be crystallized [e.g., laser crystallization ofamorphous silicon- Brendel et al. (2003), Thin Solid Films 427, Pages86-90] to be more electrochemically inert. Alternatively, an extremelythin layer of a chemically inert metal (e.g., on the order of 2 nm),such as gold or one of the platinum group metals (including, but notlimited to, PGM—ruthenium, rhodium, palladium, osmium, iridium, andplatinum), known for their chemical inertness, can be deposited in sucha manner as to prevent lateral currents. In an alternative embodiment,gold or a PGM can be patterned in an array of pads that are activated bythe photoconducting substrate below. Alternatively, or additionally, asolution of nanoparticles or microparticles, such as, but not limitedto, spin on glass, or a titania nanoparticle colloid, can be used tocoat the substrate. In a further alternative, a monolayer ofnanoparticles or microparticles can be self-assembled on the surface tocreate a uniform distribution of small electrically isolated pads.

FIG. 2 depicts schematics of two embodiments of the process ofphotoelectrochemical placement of a material at a specific location on asubstrate to create a photoelectrode substrate, according to one aspectof the present invention. In particular, FIG. 2 illustrates embodimentsof the process steps for the patterning of platinum microcontacts onamorphous silicon. In FIG. 2, in a first embodiment, a device isfabricated with plasma enhanced vapor deposition 205 (PECVD) ofamorphous silicon 210 onto conductive surface 215 on glass slide 220. Inthe embodiment shown, conductive surface 215 is indium tin oxide (ITO),but any suitable conducting surface may be used in the process of theinvention. Next, a layer of platinum 225 (Pt) is deposited 230 on top ofamorphous silicon 210. Platinum layer 225 is etched 235 using patternresist 237 and HCL HNO3 at 70 degrees C. to create patterned platinummicrocontact 240. Resist 237 is then stripped 245 with acetone, leavingpatterned platinum microcontact 240.

In a second embodiment depicted in FIG. 2, patterned micro wells arecreated first. In FIG. 2, silicon oxide dielectric layer 250 (SiO2) isdeposited 255 via PECVD onto amorphous silicon 210 on conductive surface215 on glass slide 220. Silicon oxide layer 250 is etched 260 usingpattern resists 267 and RIE, leaving patterned SiO2 270. Next, a layerof platinum 275 (Pt) is deposited 280 on top of amorphous silicon 210and patterned SiO2 270. Resists 267 are then stripped 285 with acetone,NMP, and/or sonication, leaving patterned platinum microcontact 290. Thefinished device is then subjected to plasma-assisted oxidation. Thefabrication may be made easier by eliminating the silicon oxidedielectric layer deposition, patterning, and etching steps if such aphysical barrier between electrodes is not required.

In a specific demonstrative embodiment, the device is an ITO coatedglass slide with a layer of intrinsic amorphous silicon and an array ofplatinum contacts coating the surface. The exposed silicon is oxidizedthermally or electrochemically. The surface of the device is treatedwith colloidal silica particles and sintered under nitrogen, forming aporous matrix. The matrix is treated withtriethoxysilanehydroxybutyramide. The device is treated withdimethoxytrityl chloride to protect free hydroxyls. The device is thentreated with acetic anhydride to cap any unreacted nucleophiles on thesurface. When in contact with an electrolyte solution of 10 mMhydroquinone and 50 mM tetrabutylammonium hexaflurophosphate in thefluidic (with a built-in counter electrode), the device is positivelybiased with respect to a platinum quasi-reference electrode to 1.7V, anoperating region determined by viewing the redox signature of a knowncompound such as ferrocene. Light is projected through a digitalmicromirror array for 5s (chopped at 10 Hz), after which the substrateis washed and then exposed to a cy3-phosphoramidite fluorescent dye,demonstrating the desired photoelectrochemical patterning.

A scanning electron micrograph (SEM) of a device made according to theprocess of the present invention, having patterned platinummicrocontacts 310 on amorphous silicon 320 over ITO-glass, is shown inFIG. 3. FIGS. 4A and 4B are Atomic Force Microscopy (AFM) and ScanningElectron Microscope (SEM) micrographs, respectively, of a suitableporous SiO2 reaction layer produced from sintered commercial colloidalsilica, according to one aspect of the present invention.

Since electrochemistry will still occur at the semiconducting surface,the porous layer can serve as a high surface area platform forattachment of the desired chemistry, and surface integrity of the mainsubstrate semiconductor is not as important. Alternatively, asemiconducting sol gel, such as TiO2 may be used to create a porous filmand increase the surface area in contact with the electrolyte.Alternatively, instead of a semiconducting surface with which bothelectrochemical reactions and DNA synthesis occur, a photoelectricallyactive semiconducting solution, such as silver ion or semiconductingnanoparticles, can be flowed into the chamber. In these manners,solution impedance may be altered along the light path to allow forelectrochemical reactions at the electrically active substrate surface,or for electrochemical reactions take place near the surface of the nanoor micro particles in solution rather than on the surface, so thesubstrate may be glass or another material.

Illumination of the device can be with a diode array, digitalmicromirror array, LCD or LED screen or projection system,mirror-steered laser source, transillumination through a mask,laser-scanning, or any other light source that can be spatiallymodulated or temporally modulated with the device on a moveable stage.In a typical embodiment, a DMD array is used with a 1:1 image projectionsystem, projecting a desired image on the substrate. The APO Rodagon Dis a suitable lens for a simple 1:1 projection system, but otherprojection systems can utilize other types of relay lens systems, withor without magnification or reduction. The light source may be of anywavelength that suitably creates charge carriers in the semiconductor,i.e. wavelengths of light that exceed the bandgap either of the deviceor of the device with suitable chemical sensitizers.

It is desirable to generate acids, bases, or radicals at the surface inorder to perform reactions of consequence. These reactions are typicallyspatially selective reactions that either promote or inhibit thedeprotection or addition of monomers, oligomers, or pre-synthesizedmolecules, including the tandem use of photocleavable or photogeneratedchemistries (e.g. the photoelectrochemical generation of a base tocatalyze the photocleavage of a NPPOC group). Various reagents such as,but not limited to, water, hydroquinones, anthrohydroquinones,naphthohydroquinones, phenols, or other electrochemically activecompounds capable of oxidation that yields a free proton may be used togenerate acid at the surface in the pattern. A coupled electrochemicalsystem may optionally be implemented, such as utilizing ferrocene as anelectron mediator between the electrode and the acid/base generatingcompound. Typically this has advantages, in that the surface alwaysreacts with the mediator in a known manner. Furthermore, the hydrolysisof water may also be used to generate an alkali or acid gradient at thesurface with a different bias across the semiconductor. Additionally,electro-generated bases and radicals can be used in this device. Thewell-known need for generating such reactions in a specific pattern is adesired precondition for the production of arrays of oligonucleotidessynthesized by the phosphoramidite method and arrays of peptidessynthesized by standard peptide synthesis techniques.

It will be clear to one of ordinary skill in the art that other types ofchemistries are possible with the present invention. With suitablesubstrate construction, solvents, and supporting electrolytes, almostall electrochemically controlled oxidation or reduction reactions arepossible with this light addressable system.Photoelectrochemically-generated species can remove all acid, base, andradical cleavable protecting groups. Protecting groups may also beremoved by direct electrochemical cleavage. This method is compatiblewith other biomolecule synthesis methods, including phosphotriester andH-phosphonate chemistries for DNA synthesis. Alternatively, the spatialphotoelectrochemistry can be used to selectively functionalize variouscompounds onto the surface of the device, such as, but not limited to,proteins, DNA, and other types of biomolecules. Selectivefunctionalization may be by acid-base, radical, by redox chemistry insolution, by redox chemistry directly on molecules linked to the solidsupport, or by redox chemistry applied during the coupling step to thedesired compounds, including catalysis.

Once prepared, the device can selectively release the molecule from thesubstrate for the hierarchical assembly of larger constructs. Suchhierarchical assembly schemes for genomic length DNA are disclosed in,for example, Carr, et al. (2005), U.S. Pat. App. Pub. No.US-2005-0255477 (“Method for High Fidelity Production of Long NucleicAcid Molecules”). The molecule can be released usingphotoelectrochemically generated acids, bases, and radicals to cleaveacid, base, or radical-labile linkers. Alternatively,photoelectrochemically-generated species can promote/inhibit thecleavage of molecules to be cleaved non-photoelectrochemically (i.e.chemically, bio-chemically, or by photocleavage) or vice-versa,including when the non-photoelectrochemical mechanism is either globalor spatially localized (e.g. the photoelectrochemical generation of acidto inhibit the global deprotection of a succinyl linker by a base).Alternatively, the molecule can be released by the photocleavage of aphotocleavable linker molecule if the photocleavage wavelengths are notpresent during the synthesis. In alternate embodiments, the molecule canbe released by using a photogenerated acid, base, or radicalwith/without a photoelectrochemically generated inhibitor, andvice-versa, or the molecule can be released by creating a pH gradientthat influences the interactions of biological molecules in bothone-step (e.g. promoting/inhibiting nuclease activity) and multi-stepschemes (e.g. affecting DNA hybridization that subsequently affectsenzymatic cleavage).

In order to create high-density arrays with spot sizes on the order of10 um by 10 um, it is necessary to inhibit lateral diffusion of thephotoelectrochemically generated acid. Utilizing a high viscosity gel,matrix, or wax instead, or in addition to, a solvent solution is oneapproach. The electrolyte/matrix is applied in a solution phase andcooled or cured. After the desired reaction has occurred, the matrix isstripped with a solvent or by an increase in temperature. Anotherapproach is to pattern micro wells by standard lithography techniques.In one embodiment, the patterned wells are made by depositing adielectric material (such as silicon nitride or silicon dioxide) on thesemiconductor via a spin on glass or chemical vapor depositiontechnique, applying and developing a photoresist, and etching thedielectric material with HF or reactive ion etching to expose thephotoconductive layer at the sites desired. The wells prevent lateraldiffusion.

Another approach to limit diffusion is the application of a microporousspin on glass or polymer capable of supporting phosphoramidite synthesis(i.e. resistance to multiple solvent rinses, oxidants, andelectrochemical side reactions). Thus, synthesis occurs on the porousmatrix rather than on the surface of the semiconducting device. Anothermethod involves operating the device under an AC bias, which willgenerate acid and base under the appropriate bias. The AC duty cycle canbe altered to provide the desired quenching, thus inhibiting diffusionof protons. An alternate method involves altering the duty cycle of thelight source, pulsing a specific spot and waiting for the acid to reactin a given area before creating more acid. Another method involvesaltering both the duty cycle of the light source and power supply. Inthis case, a specific pattern of light is shown when the device is underone bias, and then an alternative pattern of light is shown when thedevice is under a different or reverse bias. Yet another method involvesseparating the non-bias electrode from the substrate surface by adistance less than the inter-photoelectrode distance on the substrate.In this manner, the counteracting chemicals generated at the non-biaselectrodes will react with the reactive species before they diffusebetween the photoelectrodes.

An alternate method involves the addition of chemical scavengers to thesolution, such as pyridine, triethylamine, or any other suitable base(KOH, NaOH, etc.) for proton generation or extremely weak acids, such asammonium chloride, to scavenge free bases. In the case ofelectrochemical diffusion for reaction on alternative surface, it may benecessary to use alternating current, such as a square wave, andilluminate the semiconductor at inverse locations as the currentalternates. In this manner, acid and base can be generated atalternating spots so as to prevent lateral acid diffusion by anacid-base reaction. Additionally, in this case, a wire grid directly onthe surface of the chip, in addition to any pads for desiredelectrochemistry necessary for combinatorial synthesis, may be connectedto a global potential to generate a counteracting chemical to react withthe diffusing species of interest. In this manner, diffusion of thespecies of interest reacts with the counteracting species beforereaching any unintended surfaces.

FIG. 5 is a schematic of an example embodiment of a DMD-based spatiallymodulated illumination system with spatial modulation capabilities fordriving site-selective photoelectrochemistry, according to one aspect ofthe present invention. The system is low cost because it utilizes acommercially available digital light projector that contains its ownlight source. In FIG. 5, projector 505 is mounted on Y-Jack 510. APORhodagon D lens 515 and Gimble mount 520 are mounted on X,Z stage (1)525, which controls zoom. Gimble mount 520 corrects for astigmatism. Y,ZStage (3) 530 holds substrate 535 in vacuum chuck 540 and is mounted onX Stage (2) 545. Stage (2) 530 controls the focus of light ontosubstrate 535, while Stage (3) 530 controls the alignment of substrateelectrodes to DMD pixels. Camera 550 is mounted on X,Y,Z Stage (4) 555,which is also mounted on x Stage (2) 545 and which controls camera focusand image scroll.

FIG. 6 is a photoelectrochemical cyclic voltammogram of ferrocenedemonstrating selective photo-induced redox chemistry, according to oneaspect of the present invention. Referring to FIG. 6 to demonstrate theselective photo-response of the substrate, cyclic voltammogram 610 wastaken with 2 mM ferrocene and supporting electrolyte (50 mMtetrabutylammonium hexaflurophosphate) in acetonitrile, which clearlyshows no current generated. Second cyclic voltammogram 620 is repeatedduring exposure to light (150 mW/cm2), clearly showing the CV signatureof ferrocene. As a control, cyclic voltammogram 630 was taken duringexposure to light with the salt solution only, in which case there wasno electrochemical current generated. Thus, selective photo-inducedredox chemistry is demonstrated. The fact that the non-illuminatedcurves do not resemble a typical ferrocene CV signature, but rather thatof a resistor, indicates that the potential at the surface of theelectrode is insufficient to drive electrochemistry because thepotential drop of the semiconductor-electrolyte system is within thesemiconductor. Furthermore, the small amount of current at zero and lowbiases when the substrate is illuminated indicates that the extent ofphotoelectrochemical reactions is insignificant compared to when thesubstrate is biased at or above its threshold potential. The CV curvesof FIG. 6 also show that the photoconductor may be used as alight-sensitive electrochemical sensor, and thus the electrochemicalgeneration of reagents can be monitored real-time in some embodiments.

FIG. 7 is a fluorescence micrograph of a dye that has selectivelyreacted with functional groups that are only photoelectrochemicallydeprotected over illuminated electrodes, according to one aspect of thepresent invention.

The process of the present invention may optionally include real-timemonitoring steps. These may include, but are not limited to, monitoringthe deprotection reactions in real-time using UV absorptionspectroscopy, monitoring the generation of deprotecting agents inreal-time using a pH-sensitive dye, and electrochemically monitoring thegeneration of deprotecting agents using the photoelectrode.

The polymer arrays created by the method of the present invention havemyriad uses. In one aspect of the present invention, a light-addressablepotentiometric sensor is composed of an array created by the method ofthe present invention. In another aspect of the present invention, aphotoconductor is used to create spatial pH gradients based on thespatial modulation of light in order to influence the interactions ofbiological molecules in a spatially selective manner. In one embodiment,the biological interaction is DNA hybridization. In another aspect ofthe present invention, a photoconductor is used to create spatial pHgradients based on the spatial modulation of light in order to influencethe activity of enzymes in a spatially selective manner.

It is to be understood that the examples presented herein areillustrative of a broad range of other examples that may be constructedby combining steps involving the mechanisms detailed above. Each of thevarious embodiments described above may be combined with other describedembodiments in order to provide multiple features. Furthermore, whilethe foregoing describes a number of separate embodiments of theapparatus and method of the present invention, what has been describedherein is merely illustrative of the application of the principles ofthe present invention. Other arrangements, methods, modifications andsubstitutions by one of ordinary skill in the art are therefore alsoconsidered to be within the scope of the present invention, which is notto be limited except by the claims that follow.

What is claimed is:
 1. A method for photoelectrochemical synthesis of abiomolecule array, comprising the steps of: (a) providing asemiconductor substrate having at least one light-addressablephotoelectrode proximate to the substrate surface; (b) providing anphotoelectrochemical reaction-generating chemistry that is in contactwith the semiconductor substrate and is capable of generating reagentswhen subjected to a potential above a threshold, thephotoelectrochemical reaction-generating chemistry comprising anelectrolyte solution, matrix, gel, or solid that is suitable forphotoelectrochemical reactions at a surface; (c) applying an inputpotential to the light-addressable photoelectrode to generate chargecarriers in areas of the substrate under illumination and thereby createa patterned substrate, the applied input potential exceeding thethreshold in the presence of light and not exceeding the threshold inthe absence of light, the input potential being generated by light froma spatially-modulated light source, the light being patterned by a mask,LED, LCD, steered mirror, or digital micromirror array, wherein thecharge carriers generate electrochemical reactions via transfer ofelectrons between the semiconductor substrate and thephotoelectrochemical reaction-generating chemistry; and (d) repeatingsteps (a) to (c) until a biomolecule array of desired size issynthesized.
 2. The method of claim 1, wherein the light-addressablephotoelectrode is proximate to at least one molecule bearing at leastone chemical functional group, the chemical functional group isprotected, and the generated reagents are deprotecting.
 3. The method ofclaim 1, wherein the light-addressable photoelectrode is proximate to atleast one molecule bearing at least one chemical functional group, thechemical functional group is unprotected, and the generated reagents areactivating.
 4. The method of claim 2, wherein the protected chemicalfunctional group is located on a second parallel substrate and thephotoelectrochemical reaction-generating chemistry can diffuse towardsthe protected chemical function group on the second substrate.
 5. Themethod of claim 1, wherein the light-addressable photoelectrode isproximate to at least one molecule bearing at least one chemicalfunctional group and reagents generated by the photoelectrochemicalreaction-generating chemistry promote the removal of a protecting groupfrom the chemical functional group by another agent.
 6. The method ofclaim 1, wherein reagents generated by the photoelectrochemicalreaction-generating chemistry promote the addition of a monomer.
 7. Themethod of claim 2, wherein reagents generated by thephotoelectrochemical reaction-generating chemistry inhibit the removalof a protecting group from the chemical functional group by anotheragent.
 8. The method of claim 1, wherein reagents generated by thephotoelectrochemical reaction-generating chemistry inhibit the additionof a monomer.
 9. The method of claim 1, in which the photoelectrode isselected from the group consisting of a semiconductor, silicon, anorganic photoconductor, titanium dioxide, dye sensitized titaniumdioxide, a schottky diode, a layered structure of silicon and anothersemiconductor, a P-I-N diode, a P-N junction, and a P-N junction havinga top layer coated with an inert metal.
 10. The method of claim 1,wherein the applied input potential is selected from the groupconsisting of: the peak potential of the substrate, within +/−0.5V ofthe peak potential of the substrate, AC, AC and synchronized with theillumination source, pulsed, and pulsed and synchronized with theillumination source.
 11. The method of claim 1, wherein thephotoelectrode acts as a photoconductor that generates a potential thatis approximately linearly proportional to an applied light field and isbiased below the threshold.
 12. The method of claim 1, wherein thephotoelectrode acts as a photoconductor that is biased above the bandgapthreshold potential of the substrate so that there exists sufficientenergy for the electrons to overcome the bandgap when no light isapplied.
 13. The method of claim 1, further comprising the step ofgenerating getters for rendering neutral reagents generated by theelectrochemical reaction-generating chemistry by oppositely biasing, tothe photoelectrode, one or more adjacent photoelectrodes.
 14. The methodof claim 13, wherein the getters spatially localize the effect of anydeprotecting reagents.
 15. The method of claim 14, wherein thedeprotecting reagents are acids and the getters are bases, thedeprotecting reagents are acids and the getters are radicals, thedeprotecting reagents are bases and the getters are acids, thedeprotecting reagents are radicals and the getters are acids, or thedeprotecting reagents are radicals and the getters are radicals.
 16. Themethod of claim 1, wherein the polymer array is a DNA array and thephotoelectrochemical reactions comprise phosphoramidite synthesis. 17.The method of claim 1, wherein the synthesis is performed in a fluidiccapable of electrochemical synthesis and chemical resistance tosolvents, acids, and bases.
 18. The method of claim 1, furthercomprising the step of providing a porous reaction layer disposed on thesubstrate.
 19. The method of claim 1, wherein the light-addressablephotoelectrode is proximate to at least one molecule bearing at leastone chemical functional group that can be cleaved and thephotoelectrochemical reaction-generating chemistry is capable ofgenerating cleaving reagents.
 20. The method of claim 19, wherein thecleaving agent selectively promotes the cleavage of a molecule from asurface by another agent.
 21. The method of claim 19, wherein thecleaving agent selectively inhibits the cleavage of a molecule from asurface by another agent.
 22. The method of claim 1, wherein thephotoelectrode is a continuous photoelectrode such that differentregions of the photoelectrode may be differentially optically addressedand further comprising the step of differentially optically addressingthe continuous photoelectrode to create a spatial pattern of material.23. The method of claim 1, wherein the spatially-modulated light sourceis temporally modulated.
 24. The method of claim 1, wherein there is aone-dimensional or two-dimensional array of photoelectrodes.
 25. Themethod of claim 24, wherein the photoelectrodes are differentiallyoptically addressed to create a spatial pattern of material.
 26. Themethod of claim 2, further comprising the step of monitoring thegeneration of deprotecting agents in real-time using a pH-sensitive dye.27. The method of claim 2, further comprising the step of monitoring thedeprotection reactions in real-time using UV absorption spectroscopy.28. The method of claim 2, further comprising the step ofelectrochemically monitoring the generation of deprotecting agents usingthe photoelectrode.